U.S. Pat. No. 4,469,791 discloses novel recombinant DNA containing an amylase-coding gene which is prepared by the in vitro process of cleaving DNA derived from a bacterial donor microorganism and combining the resulting DNA fragments with a vector, which has been similarly cleaved, the vector comprising a plasmid or the DNA of a derivative of phage lambda. This recombinant DNA may be inserted into a bacterial host organism and the latter cultivated to produce the amylase. A variety of bacterial donor and bacterial host organisms are described in the U.S. patent as are a number of suitable plasmids and derivatives of phage lambda.
The use of plasmids to introduce a gene into a microorganism is a widely-used technique. Recent literature contains descriptions of a number of plasmids which have been proposed for this purpose. In particular, two articles in "Gene", published by the Elsevier Biomedical Press, 15 (1981) 43-58, by Ilkka Palva, et al, and 19 (1982) 81-87, by Ilkka Palva alone, describe the isolation of the gene coding for alpha-amylase from Bacillus amyloliquefaciens by direct shotgun cloning using B. subtilis (Bacillus subtilis) as a host. The genome of Bacillus amyloliquefaciens was partially digested with the restriction endonuclease Mbo I, and 2- to 5-kb fragments were isolated and joined to plasmid pUB110. Competent B. subtilis amylase-negative cells were transformed with the hybrid plasmids and kanamycin-resistant transformants were screened for the production of alpha-amylase.
One of the problems of using a genetically-engineered microorganism on an industrial scale is the stability of the recombinant DNA which has been introduced by the genetic engineering process. If there is a lack of stability, the recombinant DNA tends to be lost or to undergo sequence rearrangements as successive generations of the organism are produced until eventually the amylase-coding gene is no longer or only weakly expressed by descendant microorganisms.
We have now developed recombinant DNA which comprises certain amylase-coding genes described in U.S. Pat. No. 4,469,791, but which is derived from a plasmid not specifically described in that patent. The plasmid is pUB110, whih was also described in an article in the Journal of Bacteriology 1978, Vol. 134, pp. 318-329. The plasmid pUB110 comprises a gene coding for resistance to kanamycin or to analogous antibiotics inactivated by the nucleotidyl transferase enzyme. We have found that the recombinant DNA derived from this plasmid and certain amylase-coding genes may be introduced into a host-microorganism and that, particularly when the host is B. subtilis, mutant strains may be produced and cultivated which have enhanced stability and high copy numbers. The mutant microorganisms which comprise the novel recombinant DNA may be used, therefore, on an industrial basis for the production of amylase and in particular, for the production of the alpha-amylase of B. megaterium (Bacillus megaterium), an amylase possessing particularly useful commercial properties.